The present invention relates generally to methods for quantifying amounts of chemical or biochemical substances, and, more particularly, to quantitative fluorescence assays that use evanescent field excitation.
Fluorescence immunoassays or even florescence immunosensors have already been generally used for a long time, and they serve, mainly in a liquid sample matrix, to quantify an unknown amount of a specific chemical or biochemical substance. Antibodies are here selectively bound to the substance to be determined. The substance to be determined is also referred to by the expert as an antigen. In the fluorescence immunoassays, the analyte-specific antibodies are marked with a marking substance which is optically excited at a certain substance-specific wavelength xcexxcex5"khgr" and the fluorescent light with a different wave length, which is generally greater, is used with a suitable detector with evaluation of the intensity of the fluorescent light. The exploitation of the evanescent field excitation in carrying out such fluorescence immunoassays, or respectively in the fluorescence immunosensors, is already part of prior art. Thus different solutions have already been described in WO 94/27137, by R. A. Badlay, R. A. L. Drake, I. A. Shanks, F. R. S., A. M. Smith, and P. R. Stephenson in xe2x80x9cOptical Biosensors for Immunoassays; Fluorescence Capillary-Fill Devicexe2x80x9d, Phil. Trans. R. Soc. Lund. B 316, 143 to 160 (1987) and D. Christensen, S. Dyer, D. Fowers, and J. Herron, xe2x80x9cAnalysis of Exitation and Collection Geometries for Planar Waveguide Immunosensorsxe2x80x9d, Proc. SPIE-Int. Soc. Opt. Eng. Vol. 1986, Fiber Optic Sensors in Medical Diagnostics, 2 to 8 (1993).
In addition, in WO 90/05295 A1, an optical biosensor system is described. In this system, one or more samples are guided, with the use of pumps and valves, through ducts to one or more flow-through measuring cells. These flow-through measuring cells are open upwards and biomolecules can be quantitatively detected by an optical structure disposed above them. For measuring successive new samples, considerable purification outlay is consequently required, in order to avoid measuring errors. A possibly necessary preparation of such a sample generally has to be carried out externally of this system, before the actual measuring, since no elements or measures suitable for this purpose are named.
In WO 90/06503, a sensor is described in which the excitation light is directed at an appropriate angle through an optically transparent substrate onto a boundary surface to an optically transparent buffer layer. Above which an additional waveguide layer is applied, to which in turn the analytes to be determined can be bound.
The refractive index of the buffer layer is here smaller than that of the substrate and of the waveguide. At the boundary layer substrate/buffer, total reflection comes about through appropriate choice of the angle of the excitation light, and, via the evanescent field produced here, the excitation light is coupled into the waveguide situated above the buffer layer. The light coupled into the waveguide is guided via total reflection in the waveguide, and the evanescent field forming during this process is correspondingly used for fluorescence excitation.
The sample can be received in one or more cavities, the corresponding dimensions of such a cavity being only restricted to the extent that its size renders possible the transport of the samples in the cavities by means of capillary force. After the sample has been received in the cavities, no further flow or movement of the sample takes place.
The known solutions have, however, in general the disadvantage that they are only suitable for specific assay formats and an expensive structure with corresponding process management is necessary.
It is therefore an object of the invention to create a way to carry out, with a very simply constructed device, quantitative fluorescence immunoassays with different biochemical assays.
This object preferably is achieved according to the invention. Advantageous embodiments and developments of the invention will be apparent from the description of the invention provided herein.
In a device described in the not prior-published DE 196 11 025, light of at least one light source is directed at an angle xcex1 on the boundary surface of two media with differing refractive indices. Here a light source is selected which emits practically monochromatic light with a wavelength which is suitable for exciting the marking substance, in this case the fluorophore. Particularly suitable as the light source here are laser diodes, since they have a suitable beam profile and sufficient luminous efficiency, with a small constructional size and low energy consumption.
However, other light sources which emit monochromatic light can also be used.
The angle xcex1, at which the emitted light is sent to the boundary surface, determines, besides the refractive index of the material disposed in the beam path before the boundary surface, and the material adjoining same, together with the wavelength of the light, the penetration depth d for the evanescent field. The refractive index n1 of the material which is disposed in the beam path before the boundary surface must render possible total reflection at the boundary surface and should therefore be greater than the refractive index n2 of the other material disposed thereafter. The angle xcex1 is preferably so chosen that the following is true: sin(xcex1) greater than n2/n1. If this precondition is met, all the light is reflected at the boundary surface and thus total reflection is achieved. However, when this condition is met, a relatively small portion of the light penetrates through the boundary surface into the material, which is disposed in the beam path after the boundary surface, and the evanescent field is produced. Through the evanescent field, only those marking substances are optically excited which are located in the immediate proximity of the boundary surface. For carrying out the fluorescence immunoassays, the result of this is that only the marking substances of the antibodies or antigens which are bound to the surface of the boundary surface are excited. The fluorescence intensity of the light emitted by these fluorophores is thus directly proportional to the concentration of the marked antibodies or antigens bound to the surface, and, according to the biochemical assay used, proportional or inversely proportional to the antigen concentration.
Now the device described in DE 196 11 025 uses at least one light source, which emits practically monochromatic light and directs this at an angle providing the penetration depth d for the evanescent field, onto a base plate which is transparent for this light. The refractive index n1 of the base plate should be greater than 1.33. On the other side of the base plate, a cuvette-shaped receiving region is formed between a covering plate. Between the base plate and the cuvette-shaped receiving region is formed said boundary surface and the evanescent field can act with the given penetration depth d within the cuvette-shaped receiving region on marked chemical or biochemical partners, bound to the surface, of a general receptor-ligand system and excite the fluorophores used as the marking substance.
The fluorescence so caused is measured at the corresponding intensity with a detector. The detector is here disposed on the same side of the base plate as the light source.
As the detector, a single light-sensitive detector, a linear or a surface arrangement of a plurality of light-sensitive detectors can here be used.
It is also described there that it is advantageous to direct polarised light onto the sample to be determined. For this purpose, a polarizer can be arranged in the beam path of the light, following the light source.
The spacer and possibly the separating layers to be used are 0.001 to 10 mm thick, preferably 50 xcexcm, and a recess in the spacer forms the receiving region for the sample. Spacer and separating layers can preferably be a biocompatible adhesive film, which is designed to adhere on both sides.
The method according to the invention is based essentially on the fact that a defined sample volume is guided through the cuvette-shaped receiving region and there subjected to an evanescent field excitation, as has been described already. The sample volume can here be guided through the cuvette-shaped receiving region and the functional layer(s) by means of suction, pressure, and capillary forces.
In an advantageous embodiment, there is provided in a covering plate at least one inlet aperture, into which a sample container can be inserted or disposed, the aperture being so disposed in the covering plate that a connection may be produced between inlet aperture or sample container and receiving region. In addition, there is a second aperture which is also connected with the cuvette-shaped receiving region and represents an outflow.
The second aperture can also be provided in the covering plate. An external pump can be connected to this second aperture, or an internal pump can be inserted.
The invention is characterised by the fact that a relatively simply constructed basic pattern of a device according to the invention can be altered or used in the most varied form. Thus the essential elements, base plate, covering plate and spacer with cuvette-shaped receiving region, can be combined in the most varied way through functional layers, the separating layers, disposed if necessary in between yet allowing the sample to flow through. One or more of such functional layers can, however, also be arranged in the inflow region for the sample into the receiving region, an inlet aperture or a connection between a sample container which may be inserted into the inlet aperture, or the connection of inlet aperture and receiving region, forming part of this inflow region.
With the invention, the different assay formats may be carried out and thereby high- and low-molecular compounds can be equally detected. All known assay formats, such as sandwich-titration/competition and displacement formats, can be carried out.
For the case that separating layers are used between functional layers, or enclosing same, these separating layers must have corresponding openings, such that it is guaranteed that the sample volume can flow through the entire device. As separating layers, adhesive films can be used, for example, into which openings are produced by stamping.
The invention will be described in more detail below, by way of example.